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WO1998013517A1 - Procede de diagnostic d'apoptose dans des cellules - Google Patents

Procede de diagnostic d'apoptose dans des cellules Download PDF

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Publication number
WO1998013517A1
WO1998013517A1 PCT/DE1997/002204 DE9702204W WO9813517A1 WO 1998013517 A1 WO1998013517 A1 WO 1998013517A1 DE 9702204 W DE9702204 W DE 9702204W WO 9813517 A1 WO9813517 A1 WO 9813517A1
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WO
WIPO (PCT)
Prior art keywords
cells
substrate
ice
apoptosis
ced3
Prior art date
Application number
PCT/DE1997/002204
Other languages
German (de)
English (en)
Inventor
Klaus-Michael Debatin
Marek Los
Hubert Hug
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Ruprecht-Karls-Universität Heidelberg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts, Ruprecht-Karls-Universität Heidelberg filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to DE59705806T priority Critical patent/DE59705806D1/de
Priority to AT97912018T priority patent/ATE210733T1/de
Priority to EP97912018A priority patent/EP0934430B1/fr
Publication of WO1998013517A1 publication Critical patent/WO1998013517A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)

Definitions

  • the present invention relates to a method for the diagnostic detection of apoptosis in cells and a kit that can be used for this.
  • the object of the present invention is therefore to provide a method with which the extent of apoptosis in cells can be reliably estimated.
  • ICE proteases precedes the morphologically measurable apoptotic cell death and the occurrence of DNA strand breaks by several hours and is therefore an early parameter of apoptosis in otherwise intact cells.
  • ICE / Ced3 proteases is an early event in cells in which the apoptosis program has been irreversibly induced.
  • the invention is now based on the knowledge of the inventors that early events associated with the induction of intracellular apoptosis programs can be detected at the molecular level by the traceable activation of the ICE / Ced3 proteases.
  • Samples of patients who are undergoing apoptosis-inducing or apoptosis-inhibiting therapy are preferably measured ex vivo. With the method according to the invention it is therefore possible to reliably determine the effect of therapies on the basis of the induction or inhibition of apoptosis ex vivo or in vivo on intact cells.
  • the activation of the ICE / Ced3 proteases is detected by means of an imaging (optical) method, preferably a flow-cytometric method.
  • an imaging (optical) method preferably a flow-cytometric method.
  • intact cells are loaded with substrates for ICE / Ced3 proteases after permeabilization.
  • Permeabilization can take place, for example, by adding water to the cells (osmotic shock) or by the action of digitonin or other permeabilizers.
  • the permeabilization preferably takes place to such an extent that 10% of the cells subsequently appear as living in the forward / side scatter.
  • Suitable substrates for the ICE / Ced3 proteases have at least one aspartyl group.
  • the protease substrates are preferably fluorogenic for carrying out a flow cytometric measurement. Suitable Substrates are described in "Matayoshi et al., Science 247, pp. 954-958 (1 990)".
  • a fluorogenic substrate is preferably used in which 5 - [(2-aminoethyl) amino] naphthalene-1-sulfonic acid (EDANS) is the fluorogenic donor and 4- (4-dimethylaminophenylazo) benzoic acid (DABCYL) is the quenching acceptor, wherein donor and acceptor are connected to one another by means of a spacer.
  • DABCYL-YVADAPK-EDANS or DAB-CYL-DEVDAPK-EDANS are very particularly preferably used.
  • Coumarin-related substrates such as DEVD-NMA can also be used.
  • Rhodamine 110 derivatives are also suitable in which amino groups are substituted with various individual amino acids or short caspase substrate peptides.
  • the intracellular protease activity is measured optically, preferably by means of a flow cytometer, as the fluorescence of the cleaved substrate.
  • the method enables cells to be identified using surface markers in simultaneous measurement, ie for different cells it can be determined exactly which cells are still alive and which have died.
  • the advantages of the method according to the invention are that it can be carried out quickly and easily and enables sensitive detection.
  • the detection of apoptosis is possible several hours before morphologically detectable changes occur in the cells and is not influenced by the removal of apoptotic cells by the reticuloendothelial system in vivo.
  • the method according to the invention can also be carried out using a kit. This contains a protruding substrate for proteases of the ICE / Ced3 family, the substrate being in particular fluorogenic.
  • Substrates such as DAB-CYL-YVADAPK-EDANS, DABCYL-DEVDAPK-EDANS, DEVD-NMA or rhodamine 110 derivatives are very particularly preferred in which amino groups are substituted with various individual amino acids or short caspase substrate peptides.
  • Fig. 1 2D plots of the measured ICE / Ced3 activity (x-axis) against the measured autofluorescence at 660 nm (y-axis)
  • Fig. 3 Measurement of doxorubicin-induced ICE / Ced3 activity and cell death (maximum 33342 / propidium iodide) in CEM cells in a time kinetics
  • ICE / Ced3 activity (squares) can be detected 2-4 hours before the first DNA changes that show apoptotic cell death (diamonds).
  • CEM cells that were treated for 1 hour with a CD95-specific antibody (anti-APO-1; 1 ⁇ g / ml) (see Oehm, A. et al., The Journal of Biological Chemistry, Volume 267, No. 1 5 (1 992), pp. 10709-1071 5), are cultivated in cell culture medium RPMI 1 640 + 10% FCS. 10 ⁇ ⁇ ICE / Ced3 substrate DABCYL-YVADAP-EDANS (stock solution 100 mM in DMSO; stored at -70 ° C.) are added. Water (the same volume as the cell culture medium) is added to permeabilize the cells.
  • RPMI 1 640 + 10% FCS 10 ⁇ ⁇ ICE / Ced3 substrate
  • DABCYL-YVADAP-EDANS stock solution 100 mM in DMSO; stored at -70 ° C.
  • the mixture is incubated for 5 minutes at 37 ° C before ice cold 5x PBS in 50% FCS (0.25 volume of the initial volume) is added to bring the osmolarity back to normal.
  • the mixture is stored on ice and in 2 hours in Measure the flow cytometer.
  • the excitation wavelength of the substrate used is - 360 nm and the emission is measured at 488 nm.
  • untreated CEM cells are subjected to the same procedure.
  • CEM cells that were treated for 2 hours with a CD95-specific agonistic antibody (anti-APO-1; 1 ⁇ g / ml) (see Oehm, A. et al., The Journal of Biological Chemistry, volume 267, No. 1 5 (1 992), pp. 10709-1071 5), are cultivated in cell culture medium (RPMI 1 640 + 10% FCS).
  • RPMI 1 640 + 10% FCS cell culture medium
  • 100 l of ICE / Ced3 substrate DABCYL-YVADAP-EDANS stock solution 100 mM in DMSO; stored at -70 ° C.
  • digitonin in a final concentration of 0.002-0.005% (preferred concentration: 10% of the cells appear afterwards)
  • Permeabilization added as living in forward / side-scatter).
  • the mixture is incubated at 37 ° C for 5 minutes before an equal volume of ice-cold cell culture medium or PBS is added to dilute the digitonin.
  • the mixture is stored on ice and measured in the flow cytometer within 2 hours.
  • the excitation wavelength of the substrate used is - 360 nm and the emission is measured at 488 nm.
  • untreated CEM cells are subjected to the same procedure.
  • variant 2 is more effective, however the cells treated according to variant 1 provide a lower background.
  • CEM cells that have been treated with doxorubicin (1 ⁇ g / ml) are cultivated in cell culture medium (RPMI 1 640 + 10% FCS).
  • Permeabilization added as living in forward / side-scatter.
  • the mixture is incubated at 37 ° C for 5 minutes before an equal volume of ice-cold cell culture medium or PBS is added to dilute the digitonin.
  • the mixture is stored on ice and measured in the flow cytometer within 2 hours.
  • the excitation wavelength of the substrate used is - 360 nm and the emission is measured at 488 nm.
  • the recorded time kinetics is shown in FIG. 3. It can be seen from this that the ICE / Ced3 activity is detectable long before the onset of cell death and provides a reliable indication of apoptosis that has started.
  • Jurkat cells are treated for 1.5 hours with a CD95-specific antibody (anti-APO-1). They are then treated as described in variant 1 of example 1.
  • a CD95-specific antibody anti-APO-1
  • the caspase substrate D 2 rhodamine is added to a final concentration of 50 ⁇ M.
  • the caspase activity is measured in a flow cytometer (FACScan, Becton Dickinson).
  • the excitation wavelength is 488 nm.
  • the fluorescence is measured in the FL1 photomultiplier.
  • the dotted line shows the control cells that were not treated with the anti-CD95 antibody.
  • the solid line shows the fluorescence shift after antibody treatment. An increase in dead cells can only be detected by propidium iodide staining 3 hours after antibody treatment.
  • untreated Jurkat cells are subjected to the same procedure throw.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de diagnostic d'apoptose dans des cellules, selon lequel les cellules sont chargées d'un substrat pour protéases de la famille ICE/Ced3 et la réaction du substrat par la protéase est effectuée par des méthodes médicographiques. L'invention concerne également une trousse appropriée pour la mise en oeuvre de ce procédé.
PCT/DE1997/002204 1996-09-25 1997-09-25 Procede de diagnostic d'apoptose dans des cellules WO1998013517A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DE59705806T DE59705806D1 (de) 1996-09-25 1997-09-25 Verfahren zum diagnostischen nachweis von apoptose in zellen
AT97912018T ATE210733T1 (de) 1996-09-25 1997-09-25 Verfahren zum diagnostischen nachweis von apoptose in zellen
EP97912018A EP0934430B1 (fr) 1996-09-25 1997-09-25 Procede de diagnostic d'apoptose dans des cellules

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19639450A DE19639450A1 (de) 1996-09-25 1996-09-25 Verfahren zum diagnostischen Nachweis von Apoptose in Zellen
DE19639450.3 1996-09-25

Publications (1)

Publication Number Publication Date
WO1998013517A1 true WO1998013517A1 (fr) 1998-04-02

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PCT/DE1997/002204 WO1998013517A1 (fr) 1996-09-25 1997-09-25 Procede de diagnostic d'apoptose dans des cellules

Country Status (5)

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EP (1) EP0934430B1 (fr)
AT (1) ATE210733T1 (fr)
DE (2) DE19639450A1 (fr)
ES (1) ES2169851T3 (fr)
WO (1) WO1998013517A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009208A1 (fr) * 1997-08-20 1999-02-25 Coulter International Corp. Procede et reactif pour surveiller l'apoptose et la distinguer de la necrose
WO2000054049A2 (fr) * 1999-03-12 2000-09-14 Evotec Analytical Systems Gmbh Mesure de la chimiosensibilite par l'intermediaire de l'activite caspase
US6759207B2 (en) 1997-10-10 2004-07-06 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US6984718B2 (en) 1998-07-21 2006-01-10 Cytovia, Inc. Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003042703A1 (fr) * 2001-11-13 2003-05-22 Klaus-Michael Debatin Procede permettant de detecter l'induction de l'apoptose, par mesure de la liberation de substances contenues dans des organites cellulaires au moyen d'une cytometrie en flux

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0428000A1 (fr) * 1989-11-03 1991-05-22 Abbott Laboratories Substrats fluorogéniques pour la détection de l'activité prothéolytique d'enzymes
WO1993025694A1 (fr) * 1992-06-12 1993-12-23 Massachusetts Institute Of Technology Inhibiteurs de ced-3 et de proteines apparentees
WO1995034576A1 (fr) * 1994-06-10 1995-12-21 Panorama Research, Inc. Protease cytoplasmique de 24 kilodaltons activant l'apoptose par fragmentation de l'adn
WO1996013607A1 (fr) * 1994-10-28 1996-05-09 Oncoimmunin, Inc. Compositions pour la detection de proteases dans des echantillons biologiques, et procedes d'utilisation de ces compositions

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962301A (en) * 1992-06-12 1999-10-05 Massachusetts Institute Of Technology Relatedness of human interleukin-1β convertase gene to a C. elegans cell death gene, inhibitory portions of these genes and uses therefor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0428000A1 (fr) * 1989-11-03 1991-05-22 Abbott Laboratories Substrats fluorogéniques pour la détection de l'activité prothéolytique d'enzymes
WO1993025694A1 (fr) * 1992-06-12 1993-12-23 Massachusetts Institute Of Technology Inhibiteurs de ced-3 et de proteines apparentees
WO1995034576A1 (fr) * 1994-06-10 1995-12-21 Panorama Research, Inc. Protease cytoplasmique de 24 kilodaltons activant l'apoptose par fragmentation de l'adn
WO1996013607A1 (fr) * 1994-10-28 1996-05-09 Oncoimmunin, Inc. Compositions pour la detection de proteases dans des echantillons biologiques, et procedes d'utilisation de ces compositions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
E. D. MATAYOSHI ET AL.: "Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer.", SCIENCE., vol. 247, 23 February 1990 (1990-02-23), LANCASTER, PA US, pages 954 - 958, XP002056345 *
V. GAGLIARDINI ET AL.: "Prevention of vertebrate neuronal death by the crmA gene.", SCIENCE., vol. 263, 11 February 1994 (1994-02-11), LANCASTER, PA US, pages 826 - 828, XP002056344 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009208A1 (fr) * 1997-08-20 1999-02-25 Coulter International Corp. Procede et reactif pour surveiller l'apoptose et la distinguer de la necrose
US6759207B2 (en) 1997-10-10 2004-07-06 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US7270801B2 (en) 1997-10-10 2007-09-18 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US6984718B2 (en) 1998-07-21 2006-01-10 Cytovia, Inc. Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof
WO2000054049A2 (fr) * 1999-03-12 2000-09-14 Evotec Analytical Systems Gmbh Mesure de la chimiosensibilite par l'intermediaire de l'activite caspase
WO2000054049A3 (fr) * 1999-03-12 2000-12-14 Evotec Analytical Sys Gmbh Mesure de la chimiosensibilite par l'intermediaire de l'activite caspase

Also Published As

Publication number Publication date
DE19639450A1 (de) 1998-04-09
EP0934430B1 (fr) 2001-12-12
ES2169851T3 (es) 2002-07-16
EP0934430A1 (fr) 1999-08-11
ATE210733T1 (de) 2001-12-15
DE59705806D1 (de) 2002-01-24

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